Depsipeptide and congeners thereof for use as immunosuppressants

ABSTRACT

Depsipeptides and congeners thereof are disclosed having the following structure:                    
     wherein m, n, p, q, X, R 1 , R 2  and R 3  are as defined herein. These compounds, including FR901228, have activity as, for example, immunosuppressants, as well as for the prevention or treatment of patients suffering or at risk of suffering from inflammatory, autoimmune or immune system-related diseases including graft-versus-host disease and enhancement of graft/tissue survival following transplant. Also provided are methods for inhibiting lymphocyte activation, proliferation, and/or suppression of IL-2 secretion.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. Provisional ApplicationNo. 60/169,731, filed on Dec. 8, 1999 and U.S. Provisional ApplicationNo. 60/193,582, filed Mar. 30, 2000.

TECHNICAL FIELD

The present invention relates generally to depsipeptides or congenersthereof and use of the same as an immunosuppressant and, morespecifically, to the treatment and/or prevention of an immune disordersuch as autoimmune or inflammatory diseases, and for reducingimmunorejection of transplanted material, by administering to an animalan effective amount of a depsipeptide such as FR901228.

BACKGROUND OF THE INVENTION

Modulation of the immune system is desirous in a variety of contexts,from inhibiting an autoimmune response, to controlling infectiousdisease and inhibiting graft/tissue rejection. The principal approach tomitigate rejection is the pharmacological suppression of the immunesystem of the recipient. With this in mind, most immunomodulatorycompounds that are currently utilized are immunosuppressive. Since theearly 1960's the availability of these immunosuppressive agents havebeen restricted to only a few drugs. However, in the early 1980's inaddition to azathioprine and corticosteroids, cyclosporine became widelyavailable and has been the drug of choice ever since. (Kobashigawa,Trans. Proc. 30:1095-1097, 1998; Isoniemi, Ann. Chi. Gyn. 86:164-170,1997). However, the newer immunosuppressive agents are relatively few innumber and also suffer from many of the undesirable side-effectsassociated with earlier agents. While these drugs have been used toincrease survival times for transplanted organs, either as single agentsor in combination with other immunosuppressants, many are also usefulfor treating inflammatory and autoimmune diseases, delayedhypersensitivity, graft versus host diseases and similar immune systemassociated diseases.

Currently used immunosuppressive drugs include antiproliferative agents,such as methotrexate, azathioprine, and cyclophosphamide. Since thesedrugs affect mitosis and cell division, they have severe toxic effectson normal cells with high turn-over rate such as bone marrow cells andthe gastrointestinal tract lining. (Miller, Semin. Vet. Med. Surg.12(3):144-149, 1997) Accordingly, marrow depression and liver damage arecommon side effects.

Antiinflammatory compounds used to induce immunosuppression includeadrenal corticosteroids such as dexamethasone and prednisolone. Thecommon side effects observed with the use of these compounds arefrequent infections, abnormal metabolism, hypertension, and diabetes.

Other immunosuppressive compounds currently used to inhibit lymphocyteactivation and subsequent proliferation include cyclosporine, FK506, andrapamycin. These drugs inhibit either the calcium dependent phosphatasecalcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that isimportant for growth factor induced signaling (rapamycin). (Liu et al.,Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Biereret al., Curr. Opin. Immun. 5:763-773, 1993; Isoniemi (supra)).Cyclosporine and its relatives are among the most commonly usedimmunosuppressants. Cyclosporine is typically used for preventing ortreating organ rejection in kidney, liver, heart, pancreas, bone-marrow,and heart-lung transplants, as well as for the treatment of autoimmuneand inflammatory diseases such as Crohn's disease, aplastic anemia,multiple-sclerosis, myasthenia gravis, uveitis, biliary cirrhosis, etc.However, cyclosporines suffer from a small therapeutic dose window andsevere toxic effects including nephrotoxicity, hepatotoxicity,hypertension, hirsutism, cancer, and neurotoxicity. (Philip and Gerson,Clin. Lab. Med. 18(4):755-765, 1998; Hojo et al., Nature 397:530-534,1999).

Additionally, monoclonal antibodies, such as OKT3 have been used toprevent and/or treat graft rejection. Introduction of monoclonalantibodies into a patient, as with many biological materials, inducesseveral side-effects, such as rigors and dyspnea. (Richards et al.,Cancer Res. 59(9):2096-2101, 1999)

Within the context of many life-threatening diseases, organtransplantation is considered a standard treatment and, in many cases,the only alternative to death. The immune response to foreign cellsurface antigens on the graft, encoded by the major histocompatibilitycomplex (MHC) and present on all cells, generally precludes successfultransplantation of tissues and organs unless the transplant tissues comefrom a compatible donor and the normal immune response is suppressed.Other than identical twins, the best compatibility and thus, long termrates of engraftment, are achieved using MHC identical sibling donors orMHC identical unrelated cadaver donors (Strom, Clin. Asp. Autoimm.4:8-19, 1990). However, such ideal matches are difficult to achieve.Further, with the increasing need of donor organs an increasing shortageof transplanted organs currently exists. Accordingly,xenotransplantation has emerged as an area of intensive study, but facesmany hurdles with regard to rejection within the recipient animal(Kaufman et al., Annu. Rev. Immunol. 13:339-367,1995).

The host response to an organ allograft involves a complex series ofcellular interactions among T and B lymphocytes as well as macrophagesor dendritic cells that recognize and are activated by foreign antigen(Strom, supra; Cellular and Molecular Immunology, Abbas et al. (Eds.),WB Saunders Co., Penn., 1994). Co-stimulatory factors, primarilycytokines, and specific cell-cell interactions, provided by activatedaccessory cells such as macrophages or dendritic cells are essential forT-cell proliferation. These macrophages and dendritic cells eitherdirectly adhere to T-cells through specific adhesion proteins or secretecytokines that stimulate T-cells, such as IL-12 and IL-15 (Strom, In:Organ Transplantation: Current Clinical and Immunological Concepts,1989). Accessory cell-derived co-stimulatory signals stimulateactivation of interleukin-2 (IL-2) gene transcription and expression ofhigh affinity IL-2 receptors in T-cells (Pankewycz et al.,Transplantation 47:318, 1989; Cantrell et al., Science 224:1312, 1991;Williams et al., J. Immunol. 132:2330-2337, 1984). IL-2, a 15 kDaprotein, is secreted by T lymphocytes upon antigen stimulation and isrequired for normal immune responsiveness. IL-2 stimulates lymphoidcells to proliferate and differentiate by binding to IL-2 specific cellsurface receptors (IL-2R). IL-2 also initiates helper T-cell activationof cytotoxic T-cells and stimulates secretion of interferon-γ (IFN-γ)which in turn activates cytodestructive properties of macrophages(Farrar et al., J. Immunol. 126:1120-1125, 1981). Furthermore, IFN-γ andIL-4 are also important activators of MHC class II expression in thetransplanted organ, thereby further expanding the rejection cascade byenhancing the immunogenicity of the grafted organ (Pober et al., J. Exp.Med., 157:1339, 1983; Kelley et al., J. Immunol., 132:240-245, 1984).

The current model of a T-cell mediated response suggests that T-cellsare primed in the T-cell zone of secondary lymphoid organs, primarily bydendritic cells. The initial interaction requires cell to cell contactbetween antigen-loaded MHC molecules on antigen-presenting cells (APCs)and the T-cell receptor (TCR)/CD3 complex on T-cells. Engagement of theTCR/CD3 complex induces CD154 expression predominantly on CD4 T-cellsthat in turn activate the APC through CD40 engagement, leading toimproved antigen presentation (Grewal et al., Ann. Rev Immunol.16:111-135, 1998). This is caused partly by upregulation of CD80 andCD86 expression on the APC, both of which are ligands for the importantCD28 costimulatory molecule on T-cells. However, engagement of CD40 alsoleads to prolonged surface expression of MHC-antigen complexes,expression of ligands for 4-1BB and OX-40 (potent costimulatorymolecules expressed on activated T-cells). Furthermore, CD40 engagementleads to secretion of various cytokines (e.g., IL-12, IL-15, TNF-α,IL-1, IL-6, and IL-8) and chemokines (e.g., Rantes, MIP-1α, and MCP-1),all of which have important effects on both APC and T-cell activationand maturation (Mackey et al., J. Leukoc. Biol. 63:418-428, 1998).

Similar mechanisms are involved in the development of autoimmunedisease, such as type I diabetes. In humans and non-obese diabetic mice(NOD), insulin-dependent diabetes mellitus (IDDM) results from aspontaneous T-cell dependent autoimmune destruction of insulin-producingpancreatic β cells that intensifies with age. The process is preceded byinfiltration of the islets with mononuclear cells (insulitis), primarilycomposed of T lymphocytes (Bottazzo et al., J. Engl. J. Med., 113:353,1985; Miyazaki et al., Clin. Exp. Immunol., 60:622, 1985). A delicatebalance between autoaggressive T-cells and suppressor-type immunephenomena determine whether expression of autoimmunity is limited toinsulitis or progresses to IDDM. In NOD mice, a model of human IDDM,therapeutic strategies that target T-cells have been successful inpreventing IDDM (Makino et al., Exp. Anim., 29:1, 1980). These includeneonatal thymectomy, administration of cyclosporine, and infusion ofanti-pan T-cell, anti-CD4, or anti-CD25 (IL-2R) monoclonal antibodies(mAbs) (Tarui et al., Insulitis and Type I Diabetes. Lessons from theNOD Mouse, Academic Press, Tokyo, p.143, 1986). Other models includethose typically utilized for autoimmune and inflammatory disease, suchas multiple sclerosis (EAE model), rheumatoid arthritis, graft versushost disease, systemic lupus erythematosus (systemic autoimmunity—NZBxNZWF₁ model), and the like. (see, for example, Theofilopoulos and Dixon,Adv. Immunol. 37:269-389, 1985; Eisenberg et al., J. Immunol.125:1032-1036, 1980; Bonneville et al., Nature 344:163-165, 1990; Dentet al., Nature 343:714-719, 1990; Todd et al., Nature 351:542-547, 1991;Watanabe et al., Biochem Genet. 29:325-335, 1991; Morris et al., Clin.Immunol. Immunopathol. 57:263-273, 1990; Takahashi et al., Cell76:969-976, 1994; Current Protocols in Immunology, Richard Coico (Ed.),John Wiley & Sons, Inc., Chapter 15, 1998).

The aim of all rejection prevention and autoimmunity reversal strategiesis to suppress the patient's immune reactivity to the antigenic tissueor agent, with a minimum of morbidity and mortality. Accordingly, anumber of drugs are currently being used or investigated for theirimmunosuppressive properties. As discussed above, the most commonly usedimmunosuppressant is cyclosporine, but usage of cyclosporine hasnumerous side effects. Accordingly, in view of the relatively fewchoices for agents effective at immunosuppression with low toxicityprofiles and manageable side effects, there exists a need in the art foridentification of alternate immunosuppressive agents. The resentinvention meets this need and provides other related advantages.

SUMMARY OF THE INVENTION

In brief, the present invention is directed to depsipeptides andcongeners thereof (also referred to herein as “compounds”) which haveactivity as immunosuppressant agents. In one embodiment, this inventiondiscloses a method for suppressing an immune response of an animal byadministering to the animal an effective amount of a compound having thefollowing structure (I):

wherein m, n, p, q, X, Y, R₁, R₂ and R₃ are as defined below, includingpharmaceutically acceptable salts and stereoisomers thereof.

In another embodiment, novel compounds are disclosed having structure(I) above, but excluding a specific known compound (i.e., FR901228).Further embodiments include compositions containing a compound of thisinvention in combination with a pharmaceutically acceptable carrier.

In practicing the methods of the present invention, the compounds may beadministered to suppress the immune response in animals havingautoimmune disease, inflammatory disease, or graft-versus-host disease,as well as to animals having undergone an allogeneic transplant orxenogeneic transplant. Further methods of this invention includeadministration of a compound of this invention for inhibiting theproliferation of lymphocytes, for enhancing graft survival followingtransplant by administration previous to, concurrently with, orsubsequent to a transplant procedure (including allogeneic andxenogeneic transplant), for reducing IL-2 secretion from lymphocytes,for inhibiting induction of CD25 or CD154 on lymphocytes followingstimulation, and/or for inducing anergy or apoptosis in activatedT-cells while maintaining overall T-cell counts.

In another aspect the present invention provides methods for inducingimmune system tolerance to an antigen by administering to an animal adosage of a compound of structure (I). Also provided are methods forreducing secretion of TNF-α and for inhibiting the cell cycle of anactivated T-cell prior to S-phase entry by administering to a compoundof structure (I).

These and other aspects of this invention will be apparent uponreference to the following detailed description. To this end, variousreferences are set forth herein which describe in more detail certainbackground information, procedures, compounds and/or compositions, andare each hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph representing dose dependent inhibition ofperipheral blood lymphocyte (PBL) proliferation following incubationwith FR901228 and stimulation by beads having anti-CD3 and anti-CD28antibodies attached thereto.

FIG. 2 is a bar graph depicting dose dependent inhibition of peripheralblood lymphocyte (PBL) proliferation, following incubation with FR901228and stimulation with Ionomycin/PMA.

FIG. 3 is a bar graph depicting dose dependent inhibition of CD4positive T-cell proliferation, following incubation with FR901228 andstimulation by beads having anti-CD3 and anti-CD28 antibodies attachedthereto.

FIG. 4 is a bar graph depicting dose dependent inhibition of CD4positive T-cell proliferation, following incubation with FR901228 andstimulation with in vitro generated allogenic dendritic cells.

FIGS. 5A-5D are bar graphs depicting flow cytometry measurements of (A)CD154 expression, (B) CD25 expression, (C) CD69 expression, and (D) cellviability, following T-cell activation in the presence of varying levelsof FR901228.

FIG. 6 is a bar graph depicting IL-2 expression as measured by ELISA 24hours after 3×28 bead stimulation (anti-CD3 and anti-CD28 conjugatedbeads) of T-cells in the presence of varying levels of FR901228.

FIG. 7 is a bar graph depicting mean fluorescence intensity of CD4 cellsstained with CFDA-SE at day 6 following 3×28 bead stimulation or nostimulation in combination with addition of FR901228 at varying timepoints.

FIGS. 8A-8B are bar graphs depicting flow cytometry measurements of (A)CD137w, CD154, CD25, CD62L, and CD49d expression and (B) CD11a, CD134,CD26, CD54, CD95, and CD69 expression, following stimulation or nostimulation with 3×28 beads in the presence or absence of FR901228 (20ng/ml).

FIG. 9 is a bar graph depicting CD154 expression on CD4 cells followingan initial stimulation with 3×28 beads in the presence of 20 units ofIL-2 and a subsequent stimulation at day 3 to 4 with 3×28 beads, 20units of IL-2, 3×28 beads w/20 units of IL-2, or no stimulation and 20ng/ml of FR901228 (added on day three).

FIGS. 10A-10B are bar graphs depicting the presence of IL-2 and TNF-α insupernatants of PBL cells following stimulation by 3×28 beads in thepresence of FR901228 (20 ng/ml) and cyclosporine (500 ng/ml).

FIG. 11 depicts flow data of Jurkat T-cells stabily transfected with avector containing the nucleic acid sequence for green fluorescentprotein under control of the CD154 promoter 24 hours followingstimulation with 3×28 beads in the presence of 0, 10, 50, and 100 ng/mlof FR901228.

FIG. 12 depicts flow data of intracellular DNA staining of CD4 T-cellsfollowing no stimulation or stimulation with 3×28 beads for 24 hours, inthe presence of 0, 1, 10, 50, and 100 ng/ml of FR901228.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, this invention is directed to compounds, specificallyFR901228 and congeners thereof, that are useful in the context of immunesuppression. FR901228 is a depsipeptide isolated from the terrestrialbacterium Chromobacterium violaceum. It was subsequently shown thatFR901228 could inhibit transformation of Ha-ras transfected mousefibroblasts (NIH-3T3) The mutant ras protein, in which valine replacesglycine-12, is capable of inducing morphological changes in NIH-3T3cells. In these cells, the transformed phenotype is indicative ofoncogenic activation and correlates with increased tumorigenicity.Recently, a large number of drug candidates as well as natural productshave been identified that reverse this phenotype, and hence reversestransformation of tumorigenic cell lines. FR901228 is a natural productthat has been identified in this effort, and has subsequently been shownto be highly active in animal-based models. As a result, FR901228 hasreceived considerable attention as an antitumor agent.

More specifically, FR901228 is a bicyclic depsipetide (i.e., a peptidecontaining ester linkages as well as amide linkages) having thefollowing structure:

While the molecular basis for activity of FR901228 is not known, it hasbeen postulated that the disulfide bond may function as aredox-controlled conformational switch, and that the reducingenvironment inside a cell may convert this compound to the monocyclicdi-thiol form (Khan et al., J. Am. Chem. Soc. 118:7237-7238, 1996).

Manufacture of FR901228 by a fermentation process is disclosed in U.S.Pat. No. 4,977,138 assigned to Fujisawa Pharmaceutical Co., Ltd. (herebyincorporated by reference in its entirety). In that patent, fermentationof a strain of bacterium belonging to the genus Chromobacteriumviolaceum WB968 is grown in a nutrient medium containing sources ofassimilable carbon and nitrogen, and under aerobic conditions. Followingcompletion of fermentation, FR901228 is recovered and purified byconventional techniques, such as by solvent extraction, chromatographyor recrystallization.

In addition to isolation of FR901228 as a natural product, the totalsynthesis of this compound has now been reported by Khan et al. (supra).This procedure involves a 14-step process which provides FR901228 in 18%overall yield. In brief, the synthesis first involved the Carreiracatalytic asymmetric aldol reaction to yield a thiol-containingβ-hydroxy acid. The peptidic portion of the compound was assembled bystandard peptide synthesis methods. The thiol-containing β-hydroxy acidwas then coupled to the peptidic portion, and a monocyclic ringgenerated by formation of the ester (depsipeptide) linkage. The bicyclicring system of FR901228 was then formed upon conversion of the protectedthiols to a disulfide linkage.

In the practice of the present invention, FR901228 specifically, orcompounds of structure (I) generally, have been discovered to haveimmunosuppressive properties. To this end, the compounds of the presentinvention include, in addition to FR901228, compounds having thefollowing structure (I):

including pharmaceutically acceptable salts and stereoisomers thereof,

wherein

m is 1, 2, 3 or 4;

n is 0, 1, 2 or 3;

p and q are independently 1 or 2;

X is O, NH or NR;

R₁, R₂ and R₃ are the same or different and independently an amino acidside-chain moiety or an amino acid side-chain derivative; and

R is a lower chain alkyl, aryl or arylalkyl moiety.

As used herein, the term “amino acid side-chain moiety” means any aminoacid side-chain moiety present in naturally occurring proteins,including (but not limited to) the naturally occurring amino acidside-chain moieties identified in Table 1 below. Other naturallyoccurring side-chain moieties of this invention include (but are notlimited to) the side-chain moieties of phenylglycine,3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylysine, naphthylalanine,thienylalanine, γ-carboxyglutamate, phosphotyrosine, phosphoserine andglycosylated amino acids such as glycosylated serine, asparagine andthreonine.

TABLE 1 Representative Amino Acid Side Chain Moieties Amino Acid SideChain Moiety Amino Acid —H Glycine —CH₃ Alanine —CH(CH₃)₂ Valine—CH₂CH(CH₃)₂ Leucine —CH(CH₃)CH₂CH₃ Isoleucine —(CH₂)₄NH₂ Lysine—(CH₂)₃NHC(═NH)NH₂ Arginine

Histidine —CH₂COOH Aspartic acid —CH₂CH₂COOH Glutamic acid —CH₂CONH₂Asparagine —CH₂CH₂CONH₂ Glutamine

Phenylalanine

Tyrosine

Tryptophan —CH₂SH Cysteine —CH₂CH₂SCH₃ Methionine —CH₂OH Serine

Proline —CH(OH)CH₃ Threonine

When the amino acid side chain moiety is proline, it should beunderstood that the R₁, R₂ or R₃ group is joined to the adjacentnitrogen atom to form the pyrrolindinyl ring of proline. For example, inone embodiment of structure (I), wherein m, n, p and q are 1, the R₁group may be proline—that is, R₁ taken together with the adjacentnitrogen atom forms a pyrrolindinyl ring as represented by the followingstructure (II):

In addition to naturally occurring amino acid side-chain moieties, theamino acid side-chain moieties of the present invention also includevarious derivatives thereof. As used herein, an “amino acid side-chainmoiety derivative” includes modifications and/or variations to naturallyoccurring amino acid side-chain moieties, and includes embodimentswherein R₁, R₂ and/or R₃ are joined to the bicyclic ring of structure(I) by a double or triple bond. For example, the amino acid side-chainmoieties of alanine, valine, leucine, isoleucine, phenylglycine andphenylalanine may generally be classified as lower chain alkyl, aryl oraralkyl moieties. Derivatives of amino acid side-chain moieties includeother straight chain or branched, cyclic or noncyclic, substituted orunsubstituted, saturated or unsaturated lower chain alkyl, aryl oraralkyl moieties.

As used herein, “lower chain alkyl moieties” contain from 1-12 carbonatoms, “lower chain aryl moieties” contain from 6-12 carbon atoms, and“lower chain aralkyl moieties” contain from 7-12 carbon atoms. Thus, inone embodiment, the amino acid side-chain derivative is selected from aC₁₋₁₂ alkyl, a C₆₋₁₂ aryl and a C₇₋₁₂ aralkyl, and in a more preferredembodiment, from a C₁₋₇ alkyl, a C₆₋₁₀ aryl and a C₇₋₁₁ aralkyl.

When R₁, R₂ and R₃ as set forth in structure (I) are attached by eithera double or triple bond, a representative embodiment includes compoundsof structure (III) wherein R₂ is joined by a double bond:

In one embodiment of structure (III), R₂ is an unsaturated lower chainalkyl, such as ═CHCH₃, ═CHCH₂CH₃ and the like. In the case of FR901288,R₂ of structure (III) is ═CHCH₃, and m, n, p, q are each 1, X is oxygen,and the optional double bond is present (and in thetrans-configuration).

Amino acid side-chain derivatives of this invention further includesubstituted derivatives of lower chain alkyl, aryl and aralkyl moieties,wherein the substituent is selected from (but are not limited to) one ormore of the following chemical moieties: —OH, —OR, —COOH, —COOR, —CONH₂,—NH₂, —NHR, —NRR, —SH, —SR, —SO₂R, —SO₂H, —SOR, —PO₃R, —OPO₃R andhalogen (including F, Cl, Br and I), wherein each occurrence of R isindependently selected from a lower chain alkyl, aryl or aralkyl moiety.Moreover, cyclic lower chain alkyl, aryl and aralkyl moieties of thisinvention include naphthalene, as well as heterocyclic compounds such asthiophene, pyrrole, furan, imidazole, oxazole, thiazole, pyrazole,3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine, quinoline,isoquinoline and carbazole. Amino acid side-chain derivatives furtherinclude heteroalkyl derivatives of the alkyl portion of the lower chainalkyl and aralkyl moieties, including (but not limited to) alkyl andaralkyl phosphonates and silanes.

With regard p and q of structure (I), it should be understood that thesize of the peptidic-portion of the ring may be increased by theaddition of one (i.e., when either p or q is 2) or two (i.e., when bothp and q are two) amino acids moieties. For example, when p is 2 and q is1 (and X is oxygen), compounds of this invention include those of thefollowing structures (IV):

In structure (IV) above, the amino acid side chain moiety correspondingto the R₁ group of structure (I) is designated R₁′ in the first instanceand R₁″ in the second instance (since p is 2 in this embodiment) inorder to clarify that these amino acid side chain moieties may be thesame or different. In further embodiments, p is 1 and q is 2, or both pand q are 2.

In structure (I), the designation “” represents an optional double bond.When present, the double bond may be in either the cis- ortrans-configuration. In one embodiment, the double bond is in thetrans-configuration, as it is in the case of FR901288.

Depending upon the choice of the X and Y moieties, compounds of thepresent invention include esters when X is oxygen, amides when X is NHor NR. For example, when both p and q are 1, representative compounds ofthis invention include esters and amides as represented by structures(VI) and (VII), respectively:

The compounds of this invention may be prepared according to thefollowing Reaction Scheme 1:

In step (1), aldehyde 2 is prepared from dienoate 1 (wherein n=1, 2 or3) by the three-step procedure set forth by Kahn et al., J. Am. Chem.Soc. 118:7237-7238, 1996. Benzyl ester 3 is formed by Ti-(IV)-catalyzedaddition of O-benzyl, O-TMS ketene acetal to aldehyde 2 (wherein R′═Hand R″═OH, or R′═OH and R″═H). Hydrolysis of benzyl ester 3 with LiOH inMeOH/H₂O gives hydroxy acid 4.

In step (2), the peptidic portion of the compound is prepared bystandard peptide synthesis techniques starting with an appropriate aminoacid methyl ester 5. Methyl ester 5 is reacted with an N-protected aminoacid utilizing the BOP reagent to yield dipeptide 6, followed bycoupling to N-Fmoc-cysteine-(S-triphenylmethyl) when m is 1, or ananalog thereof when m is 2, to yield tripeptide 7. Tripeptide 7 is thenconverted to N-protected tetrapeptide 8, followed by deprotection of theFMOC group to yield tetrapeptide 9. In the above reaction scheme, R₁, R₂and R₃ are the same or different and independently represent an aminoacid side-chain moiety or derivative thereof as defined above. It willbe recognized that the above technique corresponds to the synthesis ofcompounds of structure (I) when p and q are both 1. In embodimentswherein one or both of p and/or q are 2, the above technique is utilizedto incorporate one or two additional amino acid groups into the peptidicportion of the compound.

In step (3), coupling of tetrapeptide 9 and hydroxy acid 4 with BOP andDIEA yields the hydroxy methyl ester 10. LiOH-mediated hydrolysis ofmethyl ester 10 provides the corresponding carboxylic acid, which maythen be converted to the monocyclic lactone intermediate 11 bycyclization with DEAD and PPh₃. Lastly, oxidation of thebis(S-triphenylmehtyl)lactone 11 with iodine in dilute MeOH solutionprovides compounds of the present invention having structure (I).

Alternatively, the compounds of this invention may also be synthesizedby the following technique:

In this embodiment, compound 4′ is made by the above procedures, andthen added, in Step (3) above, to the peptidic portion made by Step (2).The resulting intermediate is cyclized as disclosed previously to yieldcompounds of structure (I).

In the case of X being NH or NR, such compounds may be made when R′ orR″ of compound 4 are NH or NHR, respectively. In addition, when theoptional double bound of structure (I) is not present, the same reactiontechniques may be employed, but utilizing the corresponding saturatedintermediates.

The compounds of the present invention also include acid addition salts,which may be prepared by methods well known in the art, and may beformed from organic and inorganic acids. Suitable organic acids includemaleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic,trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric,gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic,glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acidsinclude hydrochloric, hydrobromic, sulfuric, phosphoric, and nitricacids. As used herein, the term “pharmaceutically acceptable salt” ofstructure (I) is intended to encompass any and all suitable salt forms.

With regard to stereoisomers, the compounds of structure (I) may havechiral centers and may occur as racemates, racemic mixtures and asindividual enantiomers or diastereomers. All such isomeric forms areincluded within the present invention, including mixtures thereof.Furthermore, some of the crystalline forms of the compounds of structure(I) may exist as polymorphs, which are included in the presentinvention. In addition, some of the compounds of structure (I) may alsoform solvates with water or other organic solvents. Such solvates aresimilarly included within the scope of this invention.

The present invention is related to the use of compounds of structure(I), in an animal subject, (preferentially a mammal and more preferablya human), for the treatment and/or prevention of immune response orimmune-mediated responses and diseases, such as the prevention ortreatment of rejection following transplantation of synthetic or organicgrafting materials, cells, organs or tissue to replace all or part ofthe function of tissues, such as heart, kidney, liver, bone marrow,skin, cornea, vessels, lung, pancreas, intestine, limb, muscle, nervetissue, duodenum, small-bowel, pancreatic-islet-cell, includingxeno-transplants, etc.; to treat or prevent graft-versus-host disease,autoimmune diseases, such as rheumatoid arthritis, systemic lupuserythematosus, thyroiditis, Hashimoto's thyroiditis, multiple sclerosis,myasthenia gravis, type I diabetes uveitis, juvenile-onset orrecent-onset diabetes mellitus, uveitis, Graves disease, psoriasis,atopic dermatitis, Crohn's disease, ulcerative colitis, vasculitis,auto-antibody mediated diseases, aplastic anemia, Evan's syndrome,autoimmune hemolytic anemia, and the like; and further to treatinfectious diseases causing abherent immune response and/or activation,such as traumatic or pathogen induced immune disregulation, includingfor example, that which are caused by hepatitis B and C infections,staphylococcus aureus infection, viral encephalitis, sepsis, parasiticdiseases wherein damage is induced by an inflammatory response (e.g.,leprosy); and to prevent or treat circulatory diseases, such asarteriosclerosis, atherosclerosis, vasculitis, polyarteritis nodosa andmyocarditis. In addition the present invention may be used toprevent/suppress an immune response associated with a gene therapytreatment, such as the introduction of foreign genes into autologouscells and expression of the encoded product.

As used herein, “an immune response” refers to the body's reaction toforeign or self antigens so that they are neutralized and/or eliminated.Cell-mediated immune response involves the production of lymphocytes bythe thymus (T-cells) in response to antigen exposure. This reaction isimportant in delayed hypersensitivity, rejection of tissue transplantsand in some infections. In humoral immune response plasma lymphocytes (Bcells) are produced in response to antigen exposure with subsequentantibody formation. This response can produce immunity orhypersensitivity. Nonspecific immune response, or inflammation is theresponse of the body's tissues and cells to injury from any source(e.g., trauma, organisms, chemical, ischemia, etc.). The initialresponse of the immune system to any threat involves vascular, chemical,and blood cell activities. Specific immune response is required wheninflammation is inadequate to cope with injury or invasion by anorganism or agent. It is directed and controlled by T and B cells.Cellular immunity refers to the T-cell response; humoral immunity is theterm previously used to refer to B-cell responses. In addition, thereference herein to CD4 or CD8 cells or the like, is meant to infer thatthe cells are positive for the CD4 or CD8 cell surface marker.

Further uses may include the treatment and/or prophylaxis of:inflammatory and hyperproliferative skin diseases and cutaneousmanifestations of immunologically mediated illnesses, such as,seborrhoeis dermatitis, angioedemas, erythemas, acne, and Alopeciaareata; various eye diseases (autoimmune and otherwise); allergicreactions, such as pollen allergies, reversible obstructive airwaydisease, which includes condition such as asthma (for example, bronchialasthma, allergic asthma, intrinsic asthma, extrinsic asthma and dustasthma), particularly chronic or inveterate asthma (for example, lateasthma and airway hyper-responsiveness), bronchitis, allergic rhinitis,and the like; inflammation of mucous and blood vessels; activity ofcertain viral infections, such as cytomegalovirus infection andEpstein-Barr virus infection.

In one embodiment, a method of treating a condition in an animal, thetreatment of which is affected or facilitated by reduction of lymphocyteproliferation and/or activation (e.g., downregulation of CD25 and/orCD154), comprising the administration of an effective amount of acompound of structure (I) is provided. The method of treating acondition in an animal, the treatment of which is facilitated byinhibition of lymphocyte proliferation and/or inhibition of activationmarkers (e.g., CD25 and CD154), and inhibition of immune function,wherein the condition may be autoimmunity, inflammation, graft/tissuerejection, or includes any of a number of indications such as thoseherein described, that are immunologically induced or exacerbated isprovided.

Accordingly, an embodiment of the invention is a method for thetreatment of autoimmune diseases. While, another embodiment of theinvention is a method for the prevention or treatment of rejection offoreign organ transplants comprising administering to a patient in needof such therapy a therapeutically effective amount of a compound of thepresent invention.

As noted above, cyclosporine is currently the leading drug used toprevent rejection of transplanted organs. The drug acts by inhibitingthe function of calcineurin in lymphocytes, thereby preventing thebody's immune system from mobilizing its vast arsenal of naturalprotecting agents to reject the transplant's foreign protein. Thoughcyclosporine is effective in fighting transplant rejection, it isnephrotoxic and is known to cause several undesirable side effects,including kidney failure, abnormal liver function, gastrointestinaldiscomfort, and induction of cancer. (Hojo et al., Nature 397:530-534,1999).

Newer, safer drugs exhibiting fewer side effects are constantly beingsearched for in the field. The present invention provides for suchimmunosuppressive agents which, without wishing to be bound to aparticular mechanism of action, appear to induce long lasting immunetolerance. FR901228 allows for partial activation of CD4 T-cells (e.g.,induction of CD69, but reduction of CD25, CD137w, CD11a, CD134, CD54,CD95, and CD154 surface expression as well as reduction of IL-2 and/orTNF-α production).

CD4 T-cells activated in the presence of FR901228 do not undergoactivation induced cell death (AICD), however they subsequently undergoapoptosis in vitro most likely due to the lack of IL-2 secretion and/orIL-2-receptor stimulation. Further, treatment of previously activatedCD4 and CD8 T-cells with compounds of the class of FR901228, such asthose depicted in structure (I), inhibits their growth and inducesapoptosis within a short time, while leaving resting T-cells apparentlyunaffected. Induction of apoptosis in responding T-cells not onlyeliminates the activated T-cells, but also induces a state of long-termimmune tolerance (Ferguson and Green, Nature Med. 5(11):1231-1232,1999). The reason for this specific tolerance is not well understood.Nonetheless, immunization with non-dividing donor cells (e.g., dendriticcells or irradiated peripheral blood lymphocytes) in the presence ofFR901228 prior to transplantation might very well confer specifictolerance to future host-versus-graft responses without further presenceof FR901228.

The term “tolerance,” as used herein, refers to a state ofnon-responsiveness of the immune system toward an antigen that it hasthe ability to react against. While not wishing to be bound to aparticular mechanism, it is believed that tolerance is induced by thecompounds of the present invention by the induction of apoptosis inactivated T-cells. For example, if T-cells are activated by an antigenand subsequently undergo apoptosis, a subsequent immune response againstthis antigen will not occur. Tolerance induction by a particularcompound may be tested by any methods and models known by those of skillin the art. In one example, primary and secondary stimulation by anantigen may be tested in the presence of the compounds of the presentinvention. In brief, an animal is inoculated with an antigen followed bya subsequent inoculation with the compound, about 2 weeks later theanimal may be inoculated with the same antigen in the absence of thecompound and secondary immune response measured. Accordingly, bothprimary and any secondary response may be measured by simple bloodanalysis. A sample that shows no secondary immune response demonstratestolerance induced by the immunosuppressive compound. Further, standardin vitro assays can be utilized to determine T-cell activation, such asCTL assays or testing for IL-2 secretion.

In addition to induction of apoptosis CD4 and CD8 T-cells induced with anumber of stimuli, including concurrent CD3 and CD28 engagement,Ionomycin/Phorbol myristic acid (PMA) stimulation, and allogeneicdendritic cell stimulation, demonstrate growth inhibition when treatedwith FR901228 either concurrently with stimulation or followingstimulation. Accordingly, such compositions vastly improve upon drugssuch as cyclosporine that inhibit CD4 T-cells very early in theactivation cascade. This causes CD4 T-cells activated in the presence ofcyclosporine to be defacto naive or non-activated cells, which do notundergo apoptosis. Thus, when cyclosporine treatment is terminated theinhibited immune response will redevelop. In other words, if CD4 cellsare in an active state in the presence of the of the compounds of thepresent invention these cells will undergo apoptosis and thus a lastingtolerance to the antigen, while when these same cells are treated withcyclosporine the immune response is only inhibited while cyclosporineremains present.

Further, FR901228 immune suppression leads to induction of anergy and/orapoptosis only in activated T-cells; thus, the general level of T-cellsand other hematopoietic cells are maintained. In contrast, bothcyclosporine and FK506 block the earliest events of T-cell activation,and thus prevent T-cells from entering a state at which they becomesusceptible to induction of apoptosis.

FR901228 has a number of features that aid its ability to act as apotent immunosuppressant. Accordingly, compounds of the class recited instructure (I) including FR901228 may have one or more of the followingcharacteristics: inhibition of growth of CD4 and CD8 T-cells induced byCD3 and CD28 engagement, Ionomycin/PMA, or allogeneic dendritic cells;inhibition of ongoing growth of CD4 and/or CD8 T-cells when added afterinitial activation; inhibition of signal transduction pathways for bothCD3/CD28 engagement and IL-2 stimulation; inhibition of the induction ofCD25, CD134, CD137w, CD154, CD11a, CD54, and CD95 on CD4 T-cells; nosignificant affect on CD69 induction or activation induceddownregulation of CD62L; reduction of IL-2 secretion from peripheralblood lymphocytes; reduction of TNF-α secretion from peripheral bloodlymphocytes; inhibition of cell cycle prior to S-phase entry foractivated T-cells; inhibition of p21cip/waf and C/EPB-α induction inactivated T-cells; inhibition of c-myc expression in activated T-cells;inhibition of IL-2-induced proliferation of activated T-cells; andinhibition of CD154 at the transcriptional level. In addition, FR901228does not affect bulk phosphorylation, as measured by phosphotyrosineWestern blots. Thus, these observations indicate that compounds ofstructure (I) affect the T-cell activation pathway at multiple points,thus allowing activation to proceed into early stages, such asphosphorylation, but preventing subsequent events of activation andproliferation.

To determine whether a particular compound is an effectiveimmnunosuppressant, a variety of methodologies known in the art may beperformed. In this regard, lymphocyte proliferation and/or activationmay be measured following contact with the compound of interest priorto, simultaneous with, or subsequent to stimulation. For example, a keyhallmark of T-cell activation and subsequent induction of proliferation(as well as being an initiator of inflammation) is the production ofIL-2, IFN-γ, CD25, CD69, or CD154, which can be measured by a variety ofmethods, including ELISA and flow cytometry. Other experimental methodscommonly employed to measure cell proliferation and cytokine secretionmay also be utilized, including a colorimetric assay employing propidiumiodide staining, MTT (3-[4,5-dimethylthiazole-2-yl],2-5-diphenyltetrazolium bromide), vital stains, CFDA-SE (5-(and6)-carboxyfluorescein diacetate-succinimidyl ester), cell count,bromodeoxyuridine incorporation, or a thymidine incorporation assay(see, e.g., Avian Dis. 43(2):172-181, 1999; Anticancer Res.17(1B):725-728, 1997; Geller, Scand. J. Immunol. 35:327-334, 1992;Levine et al., Int. J. Immun. 7(6):891-904, 1995; Hara et al., J. Exp.Med. 161:1513-1524, 1985; Harding et al., Nature 356:607-609, 1992;Linsley et al., Science 257:792-795, 1992; PCT Publication No. WO95/33823).

Methods of activating lymphocytes and thus stimulating lymphocyteproliferation are well known in the art and include stimulation in thepresence or absence of IL-2 with phytohemagglutinin (PHA), concanavalinA (ConA), anti-CD3 antibodies (in the presence or absence of anti-CD28antibodies), allogeneic cells, superantigens or ionomycin/PMA. (Paul,Fundamental Immunology, Fourth Edition, Lippincott-Raven, 1998).

A variety of in vitro and animal models exist for testing and validatingimmunosuppressive compounds of the present invention and theirapplicability to a particular immune system related disease orindication. Accordingly, one of ordinary skill in the art could easilychoose the appropriate model from those currently existing in the art.Such models include the use of NOD mice, where IDDM results from aspontaneous T-cell dependent autoimmune destruction of insulin-producingpancreatic β cells that intensifies with age (Bottazzo et al., J. Engl.J. Med., 113:353, 1985; Miyazaki et al., Clin. Exp. Immunol., 60:622,1985). In NOD mice, a model of human IDDM, therapeutic strategies thattarget T-cells have been successful in preventing IDDM (Makino et al.,Exp. Anim., 29:1, 1980). These include neonatal thymectomy,administration of cyclosporine, and infusion of anti-pan T-cell,anti-CD4, or anti-CD25 (IL-2R) monoclonal antibodies (mAbs) (Tarui etal., Insulitis and Type I Diabetes. Lessons from the NOD Mouse, AcademicPress, Tokyo, p.143, 1986). Other models include, for example, thosetypically utilized for autoimmune and inflammatory disease, such asmultiple sclerosis (EAE model), rheumatoid arthritis, graft-versus-hostdisease (transplantation models for studying graft rejection using skingraft, heart transplant, islet of Langerhans transplants, large andsmall intestine transplants, and the like), asthma models, systemiclupus erythematosus (systemic autoimmunity—NZBx NZWF₁ model), and thelike. (see, for example, Takakura et al., Exp. Hematol. 27(12):1815-821,1999; Hu et al., Immunology 98(3):379-385, 1999; Blyth et al., Am. J.Respir. Cell Mol. Biol. 14(5):425-438, 1996; Theofilopoulos and Dixon,Adv. Immunol. 37:269-389, 1985; Eisenberg et al., J. Immunol.125:1032-1036, 1980; Bonneville et al., Nature 344:163-165, 1990; Dentet al., Nature 343:714-719, 1990; Todd et al., Nature 351:542-547, 1991;Watanabe et al., Biochem Genet. 29:325-335, 1991; Morris et al., Clin.Immunol. Immunopathol. 57:263-273, 1990; Takahashi et al., Cell76:969-976, 1994; Current Protocols in Immunology, Richard Coico (Ed.),John Wiley & Sons, Inc., Chapter 15, 1998).

Subjects in need of treatment to suppress the immune system includesubjects with autoimmune disease; subjects undergoing transplantation;and subjects with cardiovascular disease; subjects with allergicreactions; and subjects with trauma or pathogenic induced immunedisregulation. Examples of autoimmune diseases include insulin-dependentdiabetes mellitus, asthma, psoriasis, ulcerative colitis, Crohn'sdisease, rheumatoid arthritis, multiple sclerosis, systemic lupuserythematosus, as well as others discussed above. Subjects with an organor cell/tissue transplant are also in need of treatment to suppress theimmune system in order to suppress or prevent organ transplantrejection. An “organ transplant” refers to transferring or“transplanting” an internal organ (e.g., heart, lung, kidney, liver,pancreas, stomach, large intestine and small intestine, and bone marrow)or external organ (e.g., skin) from a donor to a recipient, wherein thedonor is genetically distinct from the individual or animal who hasreceived the transplant. An “organ transplant” also includescross-species transplants (i.e., xenotransplants).

“An effective amount” is the dosage of compound required to achieve thedesired therapeutic and/or prophylactic effect; for example, the dosageof the compound which results in suppression of a naive or memory immuneresponse in the individual or animal, or which results in suppression ofan organ transplant rejection in the subject. A “desired therapeuticeffect and/or prophylactic effect” includes, for example, increasing thelife span or ameliorating the symptoms of an individual or animal havingor likely to have an autoimmune disease, such as asthma, psoriasis,ulcerative colitis, rheumatoid arthritis, multiple sclerosis, systemiclupus erythematosus, and the like. Examples of symptoms which can beameliorated include: hyperglycemia in diabetes; joint pain; stiffnessand immobility in rheumatoid arthritis; paralysis in multiple sclerosis;and rash and skin lesion in systemic lupus erythematosus. With respectto insulin-dependent diabetes mellitus, a “desired therapeutic orprophylactic effect” includes mitigating or preventing secondarycomplications resulting from the disease, such as vascular disorders.Suitable dosages will be dependent on the age, health and weight of therecipient, the extent of the disease, kind of concurrent treatment, ifany, frequency of treatment and the nature of the effect desired. Forexample, dosages can be from about 0.001-100 milligrams per day.Ordinarily, from 0.1 to 50 milligrams per day in one or moreapplications is effective to obtain desired results. In certainembodiments, the dosage may be adjusted such that non-activated T-cellsare maintained and substantially only activated T-cells are directed toapoptosis, anergy, and/or temporary functional non-responsiveness (i.e.,the general level of T-cells and/or other dividing cells aremaintained). In other embodiments, depsipeptide treatment achieves animmunosuppressive effect, while in other embodiments the dosage utilizeddoes not affect hematopoietic cell division, and in yet otherembodiments the dosage does not substantially affect cell divisiongenerally. However, effective dosage ranges can be readily determinedduring clinical trials and standard testing methodologies available inthe art. These dosages are the effective amounts for the prevention ortreatment of autoimmune diseases, the prevention or treatment of foreigntransplant rejection and/or related afflictions, diseases and illnesses.

A “subject” is preferably a mammal, such as a human, but can also be ananimal in need of veterinary treatment, e.g., domestic animals (e.g.,dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs,horses, chickens and the like) and laboratory animals (e.g., rats, mice,guinea pigs, and the like).

The compound can be administered alone or in conjunction with otherpharmacologically active agents, e.g., together with otherimmunosuppressive agents or together with antibiotics and/or antiviralagents. Compounds that can be coadministered include steroids (e.g.,methyl prednisolone acetate), NSAIDs and other known immunosuppressants,such as azathioprine, 15-deoxyspergualin, cyclosporine, mizoribine,mycophenolate mofetil, brequinar sodium, leflunomide, FK-506, rapamycinand related compounds. Dosages of these drugs will also vary dependingupon the condition and individual to be treated.

An effective amount of the compound can be administered by anappropriate route in a single dose or in multiple doses.

Pharmaceutically acceptable salts include both the metallic (inorganic)salts and organic salts, a list of which is given in Remington'sPharmaceutical Sciences, 17th Edition, pg. 1418 (1985). It is well knownto one skilled in the art that an appropriate salt form is chosen basedon physical and chemical stability, flowability, hydroscopicity andsolubility. As will be understood by those skilled in the art,pharmaceutically acceptable salts include, but are not limited to, saltsof inorganic acids, such as hydrochloride, sulfate, phosphate,diphosphate, hydrobromide, and nitrate or salts of an organic acid, suchas malate, maleate, fumarate, tartrate, succinate, citrate, acetate,lactate, methanesulfonate, p-toluenesulfonate or palmoate, salicylateand stearate. Similarly, pharmaceutically acceptable cations include,but are not limited to, sodium, potassium, calcium, aluminum, lithiumand ammonium (especially ammonium salts with secondary amines).Preferred salts of this invention for the reasons cited above includepotassium, sodium, calcium and ammonium salts. Also included within thescope of this invention are stereoisomers, crystal forms, hydrates andsolvates of the compounds of the present invention.

As immunosuppressants, these compounds are useful in the treatment ofautoimmune diseases, the prevention of rejection of foreign organtransplants and/or related afflictions, diseases and illnesses.

The compounds of this invention can be administered for the treatment ofautoimmune diseases, the prevention of rejection of foreign organtransplants and/or related afflictions, diseases and illnesses accordingto the invention by any means that effects contact of the activeingredient compound with the site of action in the body of awarm-blooded animal. For example, administration, can be oral, topical,including transdermal, ocular, buccal, intranasal, inhalation,intravaginal, rectal, intracisternal and parenteral. The term“parenteral” as used herein refers to modes of administration whichinclude subcutaneous, intravenous, intramuscular, intraarticularinjection or infusion, intrasternal or intraperitoneal.

The compounds can be administered by any conventional means availablefor use in conjunction with pharmaceuticals, either as individualtherapeutic agents or in a combination of therapeutic agents. They canbe administered alone, but are generally administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, troches, dragees, granules and powders, or inliquid dosage forms, such as elixirs, syrups, emulsions, dispersions andsuspensions. (See, e.g., Chan et al., Invest. New Drugs 15:195-206,1997, demonstrating the bioavailability of oral dosages of FR901228) Theactive ingredient can also be administered parenterally, in sterileliquid dosage forms, such as dispersions, suspensions or solutions.Other dosages forms that can also be used to administer the activeingredient as an ointment, cream, drops, transdermal patch or powder fortopical administration, as an opthalmic solution or suspensionformation, i.e., eye drops, for ocular administration, as an aerosolspray or powder composition for inhalation or intranasal administration,or as a cream, ointment, spray or suppository for rectal or vaginaladministration.

Gelatin capsules contain the active ingredient and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can besugar-coated or film-coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration preferably contain a watersoluble salt of the active ingredient, suitable stabilizing agents, andif necessary, buffer substances. Antioxidizing agents, such as sodiumbisulfite, sodium sulfite, or ascorbic acid, either alone or combined,are suitable stabilizing agents. Also used are citric acid and its saltsand sodium ethylenediaminetetraacetate (EDTA). In addition, parenteralsolutions can contain preservatives, such as benzalkonium chloride,methyl- or propylparaben, and chlorobutanol. In certain embodiments, thecomposition is prepared by diluting 10 mg of lyophilized depsipeptideand 20 mg of povidone, USP with 2 ml of a solution containing 20%ethanol USP in propylene glycol, USP. The solution is then dilutedfurther with 0.9% sodium chloride injection, USP to a final drugconcentration in the range of 0.02 to 5.0 mg/ml.

For administration by inhalation, the compounds of the present inventionmay be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or nebulizers. The compounds mayalso be delivered as powders which may be formulated and the powdercomposition may be inhaled with the aid of an insufflation powderinhaler device. The preferred delivery system for inhalation is ametered-dose inhalation (MDI) aerosol, which may be formulated as asuspension or solution of a compound of the present invention insuitable propellants, such as fluorocarbons or hydrocarbons.

For ocular administration, an opthalmic preparation may be formulatedwith an appropriate weight percent solution or suspension of thecompounds of the present invention in an appropriate opthalmic vehicle,such that the compound is maintained in contact with the ocular surfacefor a sufficient time period to allow the compound to treat the mucosalsurface or penetrate the corneal and internal regions of the eye.

The same dosage forms can generally be used when the compounds of thisinvention are administered stepwise or in conjunction with anothertherapeutic agent.

When drugs are administered in physical combination, the dosage form andadministration route should be selected depending on the compatibilityof the combined drugs. Thus, the term coadministration is understood toinclude the administration of the two agents concomitantly orsequentially, or alternatively as a fixed dose combination of the twoactive components.

From, the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. Further, all patents, patentapplications, journal articles, and references referred to herein areincorporated by reference in their entirety.

EXAMPLES Example I Cell Growth and Preparation

Cells isolated from human blood are grown in X-vivo media (BiowhittakerInc., Walkersville, Md.) and depending on use supplemented with orwithout 20 U/ml IL-2 (Boehringer Mannheim, Indianapolis, Ind.) andsupplemented with 5% human serum (Biowhittaker), 2 mM Glutamine (LifeTechnologies, Rockville, Md.) and 20 mM HEPES (Life Technology). JurkatE6-1 cells (ATCC, Manassas, Va.) are grown in RPMI 1640 (LifeTechnologies) supplemented with 10% fetal bovine serum (FBS)(Biowhittaker), 2 mM glutamine (Life Technologies), 2 mM penicillin(Life Technologies), and 2 mM streptomycin (Life Technologies).

Buffy coats from healthy human volunteer donors are obtained (AmericanRed Cross, Portland, Oreg.). Peripheral blood mononuclear cells (PBMC)are obtained using Lymphocyte Separation Media (ICN Pharmaceuticals,Costa Mesa, Calif.) according to the manufacturers' instructions.

Peripheral blood lymphocytes (PBL) are obtained from the PBMC fractionby incubation in a culture flask (Costar, Pittsburgh, Pa.) or withuncoated Dynabeads (Dynal, Oslo, Norway), 1×10⁸ cells/ml, 2 beads/cell,2 h at 37° C. Monocytes and macrophages are removed by adherence to theculture flask or phagocytoze the paramagnetic beads that are depleted bymagnetic cell separation according to the manufacture's instruction(Dynal). CD4 cells are purified from the PBL fraction by incubation with10 μg/ml of monoclonal antibodies against CD8 (clone G10-1), CD20 (cloneIF5), CD14 (clone F13) and CD16 (Coulter), 10⁸ cells/ml, 20 min at 4° C.After washing, cells are depleted twice with sheep anti-mouse Ig-coupleddynabeads (10⁶ cells/ml, 6 beads/cell, 20 min at 4° C.) and magneticcell separation. The purity of CD4 cells are routinely 91-95% asmeasured by flow cytometry.

Dendritic cells are generated from PBMC adhering to the culture flask(Costar), 10⁸ cells/ml, 2 h at 37° C. (without Dynabeads). Afterextensive washing, adherent cells are cultured for 7 days in mediacontaining 500 U/ml GM-CSF (Boehringer-Mannheim) and 12.5 U/ml IL-4(Boehringer-Mannheim). The resulting cell population is weakly adherentand expresses surface markers characteristic of dendritic cells(positive for HLA-DR, CD86, CD83, CD11c and negative for CD4). (note:all antibodies obtained from Becton Dickinson, CA).

The anti-CD3 mAb (OKT3) may be obtained from Ortho Biotec., (Raritan,N.J.) and the anti-CD28 mAb (9.3) may be obtained from Bristol-MyersSquibb, (Stamford, Conn.).

Example II T-cell Stimulation and Measurement of the Same

Cells are stimulated by three different methodologies 1) Dynabeads(M-450) covalently coupled to anti-CD3 (OKT-3) and anti-CD28 (9.3)antibodies (3×28 beads) according to the manufacturer's instructions(Dynal), 3 beads/cell, 2) ionomycin (Calbiochem, La Jolla, Calif.) (100ng/ml) and phorbol 12-myristate-13-acetate (PMA) (Calbiochem) (10ng/ml), 3) allogeneic dendritic cells (25,000 dendritic cells/200,000CD4 cells). All cells are stimulated at a concentration of 1×10⁶cells/ml. Cells are incubated with compounds of structure (I) (e.g.,FR901228) (NCI, Bethesda, Md.) for 1 to 2 hours prior to stimulation asoutlined above. Proliferation assays are conducted in quadruplicate in96 well flat-bottom plates. Cells are stimulated at 1×10⁶ cells/ml in afinal volume of 200 μl. Proliferation is measured by MTT assay (MTTassay kit, Chemicon International Inc., Temecula, Calif.) at day 3(stimulation method 1 and 2) or at day 6 (stimulation method 3), andresults are presented as mean value of quadruplicates. PBL cultures orpurified CD4 cell cultures are stimulated with 3×28 beads, ionomycin/PMAor allogenic dendritic cells. As demonstrated in FIGS. 1-4concentrations as low as 10 ng/ml of FR901228 completely inhibitproliferation of CD4 cells stimulated with 3×28 beads or ionomycin/PMA,whereas concentrations of 1 ng/ml of FR901228 or below have nosignificant effect (these experiments are performed by using a 2 hourincubation with FR901228 prior to stimulation). Interestingly,proliferation induced by allogenic dendritic cells is significantlyinhibited by FR901228 at concentrations as low as 1 ng/ml. Further,greater cell survival is not affected by FR901228, as assessed by sub-G1DNA measurement and integrity of the cell membrane (FIG. 5D). Lack ofcytotoxicity of FR901228 is in agreement with results described by Byrdet al., Blood 94(4):1401-1408, 1999; Bates et al., ClinicalPharmacology, Programs and Proceedings of American Society of ClinicalOncology, Abstract 693, 1999; and Chassaing et al., J. Chrmatogr. B719:169-176, 1998.

Growth and activation protocols are the same as those described above.

Example III Activation Marker Assays

The effect of FR901228 on the induction of various activation markers onCD4 cells is studied. In this regard, cells are labeled with one or moreof the following antibodies: anti-human CD4 Ab (Immunotech, Fullerton,Calif.), FITC-coupled anti-human CD11a Ab (Pharmingen), FITC-coupledanti-human CD26 Ab (Pharmingen), FITC-coupled anti-human CD49d Ab(Coulter), FITC-coupled anti-human CD54 Ab (Pharmingen and BectonDickinson), FITC-coupled anti-human CD95 Ab (Pharmingen), FITC-coupledanti-human CD134 Ab (Pharmingen), FITC-coupled anti-human CD25 Ab(Becton Dickinson, Fullerton, Calif.), FITC-coupled anti-human CD69 Ab(Becton Dickinson), FITC- or PE-coupled anti-human CD154 Ab (BectonDickinson), or FITC- or PE-coupled IgGI isotype control Ab. Cells, 2×10⁵are labeled for 20 minutes at 4° C. with 2 μl of each antibody in afinal volume of 30 μl, washed and resuspended in 1% parformaldehyde(Sigma, St. Louis, Mo.). Interestingly, CD25 and CD154 expression isstrongly suppressed after 3×28 bead stimulation at concentrations as lowas 10 ng/ml FR901228 (FIGS. 5A and 5B). In contrast, CD69 induction isnot affected by 100 ng/ml FR901228 (FIG. 5C). In addition, FIGS. 8A-8Bdemonstrate inhibition of the induction of CD25, CD134, CD137w, CD154,CD11a, CD54, and CD95 on CD4 T-cells with no significant affect on CD69induction or activation induced downregulation of CD62L (FIGS. 8A-8B).This suggests that FR901228 imposes a specific, however not complete,inhibition of proximal CD4 cell activation. Further, greater cellviability is not significantly affected by concentrations up to 100ng/ml of FR901228 as measured by propidium iodide (PI) exclusion usingstandard flow cytometric procedures (see Dengler et al., AnticancerDrugs. 6(4):522-532, 1995).

FR901228 inhibition cannot be bypassed by ionomycin/PMA activation,implying that FR901228 inhibition is downstream of the rise inintracellular calcium observed immediately after CD3 stimulation of CD4cells.

Example IV IL-2 and TNF-α Assays

Cells are prepared as described above. Supernatants from cellsstimulated 24 h are subjected to an IL-2 or TNF-α enzyme linkedimmunosorbant assay (ELISA) according to the manufacturer's instructions(Biosource International, Sunnyvale, Calif.).

In an alternative assay, IL-2 is measured by intracellular staining ofCD4 T-cells using flow cytometry. For intracellular labeling of IL-2 orIFN-γ, cells are first incubated with 1 μg/ml Monensin (Calbiochem) for4 hours prior to assay. The cells are subsequently stained for surfaceproteins as described above, fixed and permeabilized using BectonDickinson intracellular staining-kit, labeled with PE-coupled anti-humanIL-2 Ab and FITC coupled anti-human IFN-γ or the corresponding controlAbs as described by the manufacturer. Data acquisition and flowcytometric analysis is performed on a Becton Dickinson FACSCalibur flowcytometer using Cellquest software following the manufacturer's protocol(Becton Dickinson).

To study the mechanism behind FR901228 inhibition of CD4 cellproliferation, IL-2 and TNF-α production by activated CD4 cells wasanalyzed. FR901228 at concentrations as low as 10 ng/ml markedlyinhibits IL-2 production measured by ELISA after 24 h of 3×28 (anti-CD3and anti-CD28 antibody coupled beads) bead stimulation of purified CD4cells (FIG. 6). A similar inhibition was observed using intracellularIL-2 measurement (not shown). However, diminished IL-2 production is notsolely responsible for the lack of T-cell proliferation, as addition of100 U/ml IL-2 (Boehringer Manheim) will not restore proliferation. Theobservation that FR901228 inhibits IL-2 production contrasts withprevious results reported by Wang et al. who reported that FR901228 didnot inhibit CD3-induced IL-2 production of the A1.1 T-cell hybridoma(Oncogene 17(12):1503-1508, 1998). The reason for this difference iscurrently unknown.

In further experiments, both IL-2 and TNF-α were measured by ELISAfollowing 24 hours of stimulation of peripheral blood lymphocytes (PBL)with 3×28 beads in the presence or absence of 20 ng/ml of FR901228 and500 ng/ml of cyclosporine (FIGS. 10A and 10B).

Example V Proliferation Inhibition Assays

Peripheral blood lymphocytes are prepared as described above, stimulatedwith 3×28 beads and stained with carboxyfluorescein diacetatesuccinimidyl ester (CFDA-SE) at day 6 post stimulation. Cell stimulationwas carried out as indicated above and the cells were washed twice withPBS and resuspended in media and incubated with CFDA-SE (MolecularProbes, OR.). After about 10 minutes of staining, the cells are washedwith media and FPS and dye incorporation was measured. FR901228 wasadded at various time points post-stimulation, at time 0 or at 24, 72,and 120 hours post-stimulation. FIG. 7 depicts flow data generated byCFDA-SE staining. The y-axis sets forth mean fluoresence intensity,which relates to the inverse of cell growth. Accordingly, the dataindicates that cell growth of activated T-cells is inhibited by FR901228either by contacting the cells with FR901228 prior to, concurrentlywith, or subsequent to stimulation with 3×28 beads.

Example VI CD154 Expression on CD4 Cells

The effect of FR901228 on the induction of the CD154 activation markeron CD4 cells is studied. In this regard, cells are labeled withFITC-coupled anti-human CD4 Ab (Immunotech, Fullerton, Calif.), PE-coupled anti-human CD154 Ab (Becton Dickinson, Fullerton, Calif.), orFITC- or PE-coupled IgG1 isotype control Ab. Cells, 2×10⁵ are labeledfor 20 minutes at 4° C. with 2 μl of each antibody in a final volume of30 μl, washed and resuspended in 1% parformaldehyde (Sigma, St. Louis,Mo.). The cells are stimulated for four days in the presence of 3×28beads and/or 20 units/ml IL-2 or an anti-IL-2 antibody at day three,FR901228 is added to the culture at a concentration of 20 ng/ml. On day4, mean fluorescence intensity is measured by flow cytometry. Asdepicted in FIG. 9, the presence of absence of IL-2 did not compensatefor the suppression of CD154 expression induced by FR901228.

Example VII Repression of CD 154 at the Transcriptional Level

In this experiment Jurkat T-cells were stabily transfected with a vectorconstruct (p-EGFP1, Clonetech) wherein the nucleotide sequence encodinggreen fluorescent protein was operably linked to the CD154 promotercloned from Jurkat cells. Following selection of cells containing thevector of interest, the cells were subjected to stimulation for 24 hourswith 3×28 beads in the presence of varying concentrations (0 to 100ng/ml) of FR901228. Fluorescence was then detected using flow cytometry.As is demonstrated by FIG. 11, only cells having 0 ng/ml of FR901228showed induction of GFP expression beyond those cells having nostimulation.

In separate experiments, RT-PCR of the CD154 transcript was carried outafter 18 hours of stimulation with 3×28 beads and various amounts ofFR901228. These experiments demonstrated reduced levels of CD154expression with increasing amounts of FR901228 (data not shown).Experiments were also conducted using CD4 cells incubated withanti-sense constructs of c-myc. After stimulation by 3×28 beads fortwenty-four hours, the anti-sense c-myc construct reduced CD154expression as measured by flow cytometry by about 50% as compared tocontrols including: no construct, sense or transfection with a scrambledc-myc construct (data not shown). Accordingly, FR901228 and likecompounds may target the activity of c-myc and its induction of CD154 asone mechanism of immune suppression.

Example VIII Inhibition of Cell Cycle Prior to S-Phase Entry

Unstimulated and stimulated (3×28 bead stimulation) CD4 cells wereincubated with varying concentrations FR901228 and the intracellular DNAcontent was measured by staining with propidium iodide (PI) usingstandard procedures. In brief, the cells were stained with a mixture of1 μg/ml PI and 0.03% saponin in PBS for about 20 to 30 minutes. Asindicated by FIG. 12, DNA synthesis does not take place innon-stimulated cells or in stimulated cells incubated with 10 to 100ng/ml of FR901228. Accordingly, FR901228 appears to inhibit the cellcycle of activated T-cells prior to entry of S-phase.

Example IX Radioisotope T-Cell Proliferation Assays

Peripheral blood mononuclear cells (PBMC) from healthy donors areseparated by density centrifugation with ficoll-hypaque (LSM, OrganonTeknika, Durham, N.C.). After washing, the PBMC with complete media(RPMI 1640 medium with 5% human serum, 100 mM glutamine, 1 mM sodiumpyruvate, 0.1 mM non-essential amino acid, 2 mM Penicillin (LifeTechnologies), and 2 mM Streptomycin (Life Technologies), they are thenirradiated at 7,500 RADS, and resuspended at 4-4.5×10⁶ cells/ml incomplete media. Another aliquot of PBMC are rosetted withneuraminidase-treated sheep red blood cells (SRBC). After anothercentrifugation with LSM, the SRBC of these rosetted T-cells are thenlysed with ammonium chloride lysing buffer (Life Technologies). Afterwashing twice with complete media, these purified T-cells are alsoresuspended at 2-2.5×10⁶ cells/ml in complete media. The variousdilutions of the test compound are added in triplicate at 50 μl/wellinto a 96-well flat-bottom microculture plate (Costar, Cambridge,Mass.). The T-cell suspension is then immediately distributed into thewells at 100 μl/well. After incubating the cells with the test compoundfor 30 min. at 37° C. in a humidified atmosphere of 5% CO₂-95% air,anti-CD3 Ab (OKT-3, Ortho Diagnostic, New Jersey) is added per well(final conc. of 10 ng/ml), followed by 50 μl of the irradiated PBMC. Theculture plate is then incubated at 37° C. in a humidified atmosphere of5% CO₂-95% air for 72 hours. The proliferation of T lymphocytes isassessed by measurement of tritiated (³H) thymidine incorporation.During the last 18-24 hours of culture, the cells are pulse-labeled with2 μCi/well of tritiated thymidine (NEN, Cambridge, Mass.). The culturesare harvested on glass fiber filters using a multiple sample harvester(MACH-II, Wallace, Gaithersburg, Md.). Radioactivity of filter discscorresponding to individual wells is measured by standard liquidscintillation counting methods (Betaplate Scint Counter, Wallace). Meancounts per minute of replicate wells are calculated and the results areexpressed as concentration of compound required to inhibit tritiatedthymidine incorporation of T-cells by 50%.

Example X Prevention and/or Delay of Diabetes Onset in NOD or NODSCIDMice

This Example illustrates the ability of a representative depsipeptidecompound FR901228 to prevent or delay diabetes onset in NOD and NODSCIDmice.

FR901228 was dissolved in 10% DMSO in PBS and were administered i.p. toNOD or NODSCID mice every other day. Controls contained only the DMSOexcipient (10% DMSO in PBS). Given that, NODSCID mice do notspontaneously develop diabetes, 30×10⁶ NOD spleen cells from a 10 weekold NOD mouse were injected into each NODSCID mouse (i.v.) at 4 weeks ofage to induce diabetes. 0.5 mg/kg of FR901228 was administered at eachtreatment to twenty animals (ten NOD animals and ten NODSCID animals).Every two weeks the number of mice in each treatment group that hadbecome diabetic was evaluated by measuring blood glucose levels using aglucometer at bi-weekly intervals. A reading of more than 200 mg/dl ofblood glucose on two consecutive observations was considered indicativeof frank diabetes. Average glucose numbers for these groups arepresented in Table 2 below.

As shown in Table 2, in NODSCID mice, onset of frank diabetes wasdelayed by at least two weeks in those mice treated with the compoundwhen compared to those mice that were treated with excipient only.Surprisingly, the results of the NOD mice were even more dramatic, inthat no NOD mouse treated with the compound developed frank diabetesduring the 18 week study, while 8 of 10 excipient treated mice developedfrank diabetes in the same timeframe. Clearly, this data demonstrates animmunosuppressive effect in the delay of phenotypical diabetes onset.

TABLE 2A FR901228 injected NOD SCID mice versus injection with DMSOvehicle recipient donor age (wks) age injection Cage @ (wks.) genot sexdate card ID genot sex origine injection DOB 10 NOD 3/27/00 30 ×10{circumflex over ( )}6 518374 2659 NODSCID F BSLC 4 rec'd NOD spleencells FR901228 — — — — 2660 — — — — 22-Mar — — — — 2661 — — — — @ — — —— 2662 — — — — 4-6 wks. — — — — 2663 — — — — — — — — — 518375 2654 — — —— — — — — — 2655 — — — — — — — — — 2656 — — — — — — — — — 2657 — — — — —— — — — 2658 — — — — — 10 NOD 3/27/00 30 × 10{circumflex over ( )}6518376 2675 NODSCID F BLSC 4 rec'd NOD spleen cells (DMSO Vehicle only)— — — — 2676 — — — — 22-Mar — — — — 2677 — — — — @ — — — — 2678 — — — —4-6 wks. — — — — 2679 — — — — — — — — — 518377 2381 — — — — — — — — —2382 — — — — — — — — — 2383 — — — — — — — — — 2384 — — — — — — — — —2385 — — — — — donor 5/9 5/23 6/6 6/20 7/4 7/18 8/1 8/15 age age (wks.)(wks.) genot sex 6 8 10 12 14 16 18 20 10 NOD — — 482 High 536 Sac'd byB.S. — — — 547 526 566 402 Sac'd by B.S. — — — — 343 596 Dead — — — —317 568 Dead — — — — 299 539 494 Sac'd by B.S. — — — — 412 338 Dead — —— — 441 240 Dead — — 261 527 494 Dead — — — — 399 High Dead — — — — 285576 558 571 Dead 10 NOD 383 Dead 5/25 — — — 439 428 Dead 6/13 — — — 254377 High 337 Sac'd 7/12 by B.S. — — — 223 448 High 523 Sac'd 7/12 byB.S. — — 287 High High Dead 6/8 — — — — 311 Sac'd by B.S. — — — 447 516″ — — — 324 405 ″ — — — — 456 ″ — — — 423 463 ″

TABLE 2B FR901228 injected NOD mice versus injection with DMSO vehiclerecipient age (wks) 5/9 5/23 6/6 6/20 7/4 7/18 8/1 8/15 @ wks. Posttransfer Cage card ID genot. sex origine injection DOB 6 8 10 12 14 1618 20 FR901228 518450 2664 NOD F BSLC N/A rec'd — — — — — — — Treated2665 — — — — 23-Mar — — — — — — — 2666 — — — — @ — — — — — — — 4 wks.2667 — — — — — — — — — — — — 2668 — — — — — — — — — — — — 518449 2669 —— — — — — — — — — — — 2670 — — — — — — — — — — — — 2671 — — — — — — — —— — — — 2672 — — — — — — — — — — — — 2674 — — — — — — — — — — — —Controls with 518448 2386 NOD F BSLC N/A rec'd — — — — 238 423 452 10%DMSO 2387 — — — — 23-Mar — — — — — — — Vehicle 2388 — — — — @ — 302 513Sac'd by B.S. 2389 — — — — 4 wks. — — — — — — 310 2390 — — — — — — — —545 554 Sac'd 7/12 by B.S. 518451 2391 — — — — — — — — — — — — 2392 — —— — — — — — — 308 455 504 2393 — — — — — — — — — — — 466 2394 — — — — —— 390 391 Sac'd by B.S. 2395 — — — — — — — — — 207 354 126

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention.

What is claimed is:
 1. A compound having the following structure:

or a pharmaceutically acceptable salt or stereoisomers thereof, whereinm is 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2;X is O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety; with the proviso that the compound is not FR901228.
 2. Thecompound of claim 1 wherein m is 1 and n is 0, 2 or
 3. 3. The compoundof claim 1 wherein m is 2, 3 or 4 and n is
 1. 4. The compound of claim 1wherein one of p or q is 2, or both p and q are
 2. 5. The compound ofclaim 1 wherein X is O.
 6. The compound of claim 1 wherein X is NH orNR.
 7. The compound of claim 1 wherein R₁ and R₃ are amino acid sidechain moieties.
 8. The compound of claim 1 wherein R₂ is an amino acidside chain derivative.
 9. A composition comprising a compound of claim 1and a pharmaceutically acceptable carrier.
 10. A method of preventing orsuppressing an immune response or immune mediated response of an animal,comprising administering to said animal an effective amount of acompound having the following structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 11. The method of claim 10 wherein said animal suffers from oris at risk of suffering from an autoimmune disease.
 12. The method ofclaim 10 wherein said animal suffers from or is at risk of sufferingfrom an inflammatory disease.
 13. The method of claim 10 wherein saidanimal suffers from or is at risk of suffering from graft-versus-hostdisease.
 14. The method of claim 10 wherein said animal will undergo orhas undergone an allogeneic transplant.
 15. The method of claim 10wherein said animal will undergo or has undergone an xenogeneictransplant.
 16. A method for inhibiting the proliferation oflymphocytes, comprising administering to an animal an effective amountof a compound having the following structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 17. A method for enhancing graft survival following transplant,comprising administering to an animal previous to, concurrently with, orsubsequent to a transplant procedure an effective amount of a compoundhaving the following structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 18. The method of claim 17 wherein said transplant is anallogeneic transplant.
 19. The method of claim 17 wherein saidtransplant is an xenogeneic transplant.
 20. A method for reducinginterleukin-2 secretion from lymphocytes, comprising administering to ananimal an effective amount of a compound having the following structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 21. A method for inhibiting induction of CD25 or CD154 onlymphocytes following lymphocyte stimulation, comprising administeringto an animal an effective amount of a compound having the followingstructure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 22. A method for inducing anergy or apoptosis in activatedT-cells while maintaining overall T-cell counts, comprisingadministering to an animal a dosage of a compound having the followingstructure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 23. A method for inducing immune system tolerance to an antigen,comprising administering to an animal a dosage of a compound having thefollowing structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 24. A method for reducing secretion of tumor necrosisfactor-alpha, comprising administering to an animal a dosage of acompound having the following structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl aryl or arylalkylmoiety.
 25. A method for inhibiting the cell cycle of an activatedT-cell prior to S-phase entry, comprising administering to an animal adosage of a compound having the following structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein mis 1, 2, 3 or 4; n is 0, 1, 2 or 3; p and q are independently 1 or 2; Xis O, NH or NR; R₁, R₂ and R₃ are the same or different andindependently an amino acid side-chain moiety or an amino acidside-chain derivative; and R is a lower chain alkyl, aryl or arylalkylmoiety.
 26. The method of any one of claims 10, 16, 17, 20, 21, 22, 23,24, or 25 wherein m is
 1. 27. The method of any one of claims 10, 16,17, 20, 21, 22, 23, 24, or 25 wherein m is 2, 3 or
 4. 28. The method ofany one of claims 10, 16, 17, 20, 21, 22, 23, 24, or 25 wherein n is 1.29. The method of any one of claims 10, 16, 17, 20, 21, 22, 23, 24, or25 wherein n is 0, 2 or
 3. 30. The method of any one of claims 10, 16,17, 20, 21, 22, 23, 24, or 25 wherein p and q are both
 1. 31. The methodof any one of claims 10, 16, 17, 20, 21, 22, 23, 24, or 25 wherein oneof p or q is 2, or both p and q are
 2. 32. The method of any one ofclaims 10, 16, 17, 20, 21, 22, 23, 24, or 25 wherein X is O.
 33. Themethod of any one of claims 10, 16, 17, 20, 21, 22, 23, 24, or 25wherein X is NH or NR.
 34. The method of any one of claims 10, 16, 17,20, 21, 22, 23, 24, or 25 wherein R₁ and R₃ are the same or differentand independently an amino acid side chain moiety.
 35. The method ofclaim 34 wherein R₁ and R₃ are both —CH(CH₃)₂.
 36. The method of any oneof claims 10, 16, 17, 20, 21, 22, 23, 24, or 25 wherein R₂ is an aminoacid side chain derivative.
 37. The method of claim 36 wherein R₂ is═CHCH₃.
 38. The method of any one of claims 10, 16, 17, 20, 21, 22, 23,24, or 25 wherein the optional double bond is present.
 39. The method ofclaim 38 wherein the double bond is in the trans-configuration.
 40. Themethod of any one of claims 10, 16, 17, 20, 21, 22, 23, 24, or 25